Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders

Definitions

• This invention relates to the stability of a wide range of molecules.
• the invention relates to the stability of proteins and other biological molecules which exhibit formation of dimers or higher molecular weight species.
• the invention also relates to the stability of a wide range of molecules, ranging from small molecules to complex supramolecular systems, in particular to the stability of such molecules wherein a hydrolysis of a bond between two conjugated parts of the molecule or the system is a problem.
• the invention relates to stability of molecules in aqueous systems, for example in an aqueous solution, in aqueous gel form or in non-liquid state such as solid state where free or bound water is present e.g. in frozen condition or following partial removal of water such as by drying or freeze- drying.
• HMWS dimers or higher molecular weight species
• aggregation can be either reversible or irreversible, depending on the nature of the interactions between the protein molecules.
• a number of different types of non-covalent interactions can be engaged in protein aggregation, such as ionic interactions between positively and negatively charged parts of the protein molecules, or hydrophobic interactions between hydrophobic patches at the protein surface. In rare cases, even covalent interactions such as disulphide bonds can facilitate protein aggregation. Whilst the different types of interactions can combine, it is typical that one particular type is the dominant force in the process of HMWS formation.
• HMWS HMWS
• hydrophobic interactions HMWS
• the conditions that drive the formation of HMWS forward vary depending on the dominant interactions involved. Consequently, different conditions can be employed to minimise the rate of HMWS formation of different proteins.
• HMWS can be measured by various techniques such as size- exclusion chromatography. Formation of large aggregates can be followed by various light-scattering techniques or microscopic or visual assessment.
• Another particular stability problem of many different classes of molecules is cleavage of a bond between two conjugated parts of the molecule or the system.
• undesirable processes include cleavage of a polysaccharide moiety from a carrier protein in a number of polysaccharide-based vaccines (e.g. Haemophilus influenzae b vaccine) or a cleavage between key domains of fusion proteins (e.g. Etanercept). Acid or base hydrolysis is typically the mechanism of such degradation processes.
• Hydrolysis is a chemical reaction during which a water molecule is split into hydrogen and hydroxide ions which go on to participate in cleavage of a particular covalent bond. Hydrolysis requires the presence of water and is known to be a pH-dependent process. However, proton transfer from molecules can also be involved in the mechanism of hydrolytic cleavage.
• the present invention is based on the discovery of several desirable parameters of aqueous formulations of small molecules, macromolecules such as proteins and supramolecular systems.
• Application of the invention results in an improvement of stability, potentially substantial, of such molecules or systems.
• application of the invention results in desirable reduction of formation of dimers and HMWS during storage.
• application of the invention results in desirable reduction of the rate of destabilising hydrolytic processes.
• small molecule is used herein to encompass a molecule of any chemical structure with a molecular weight between 50 - 2000 Da.
• macromolecule is used herein to encompass a molecule of any chemical structure with a molecular weight higher than 2000 Da. Macromolecules will typically be of polymeric nature, but the invention is not limited to the polymeric macromolecules.
• protein is used herein to encompass molecules or molecular complexes consisting of a single polypeptide, molecules or molecular complexes comprising two or more polypeptides and molecules or molecular complexes comprising one or more polypeptides together with one or more non-polypeptide moieties such as prosthetic groups, cofactors etc.
• polypeptide is intended to encompass polypeptides comprising covalently linked non-amino acid moieties such as glycosylated polypeptides, lipoproteins etc.
• upramolecular systems is used herein to encompass any system made up of a discrete number of assembled molecular subunits or components.
• substance used in therapy is used herein to encompass any substance which is developed with the intention to be used in clinical trials or to be approved as part of a medical device or as a drug product.
• high molecular weight species is used herein to encompass any species formed by aggregation of the native form of a species, such as a protein. The term encompasses both soluble and insoluble aggregated forms.
• displaced buffer is used herein to encompass any additive present in a composition of specified pH which is capable of exchanging protons and has pK a value(s) at least 1 unit more or less than the pH of the composition at the intended temperature range of storage of the composition.
• displaced buffers are described in WO2008/084237, the content of which is incorporated herein by reference. In that specification, the importance of, and the distinction between, conventional and displaced buffers is described.
• ionic strength is used herein as the following function of the concentration of all ions in a solution: where C x is molar concentration of ion x (mol L "3 ), Z x is the absolute value of the charge of ion C x . The sum covers all ions (n) present in the solution.
• HMWS Many proteins and other biological molecules undergo the process of aggregation, i.e. the formation of HMWS, during storage, especially in aqueous solutions. Aggregation is typically facilitated by non-covalent interactions such as charge-charge interactions or hydrophobic interactions between amino acid residues at the surface of individual protein molecules. Both the charge and the hydrophobicity of amino acid side chains are pH dependent. For example, histidine residue (pK a about 6.1) exists predominantly in the charged form at pH ⁇ 6.1 and predominantly in the uncharged form at pH > 6.1 , the uncharged form being considerably more hydrophobic than the charged one. Consequently, the tendency of proteins and other biological molecules to aggregate is also dependent on pH.
• a preferred feature of the present invention in relation to reducing the rate of HMWS formation of proteins and other biological molecules is in combining the following formulation features in the formulation of a protein or other biological molecules or supramolecular systems:
• ionic strength of the formulation is kept minimal, such as less than 40 mM, preferably less than 20 mM, most preferably less than 1O mM.
• amphiphilic compound which can be usefully employed in the protein compositions according to the present invention is benzoic acid, particularly its ionic form (benzoate ion).
• the formulation is substantially free of a conventional buffer, i.e a compound with pK a within 1 unit of the pH of the composition at the intended temperature range of storage of the composition, and comprises one or more additives (displaced buffers) which are capable of exchanging protons with the biological molecule and have pK a values at least 1 unit more or less than the pH of the composition at the intended temperature range of storage of the composition; the art of applying displaced buffers to formulations of biologicals is described in PCT/BG2007/000082.
• the rate of the undesirable HMWS formation can be reduced substantially.
• the formulation is kept at a pH at which the rate of HMWS formation is minimal.
• Optimal pH can be established experimentally. However, the invention is applicable at pH away for such pH optimum.
• the invention is particularly applicable to stabilising substances used in therapy.
• the formation of dimers or HMWS is very likely to involve hydrophobic interactions. It means that hydrophobic regions at the surface of two or more protein molecules interact and engage in non-covalent binding interactions. This leads to gradual aggregation.
• hydrophobic bonds is known to be thermodynamically driven by increase in entropy of the system by eliminating unfavourable interactions between the hydrophobic regions and the surrounding aqueous environment. Importantly, the increase in entropy will be even higher if there is a high concentration of charged species present in the aqueous environment.
• HMWS if facilitated mainly by hydrophobic interactions, is likely to proceed more readily at high ionic strength than at low ionic strength. This is particularly the case if the protein is not kept at an optimal pH with respect to minimal aggregation.
• a typical formulation of a therapeutic protein or other biological molecule contains a buffer (for example phosphate, histidine or citrate) and one or more of the following excipients: tonicity modifiers (for example inorganic salts or amino acids), surfactants (for example Polysorbate 80) and sugars or polyalcohols (for example sucrose or mannitol).
• tonicity modifiers for example inorganic salts or amino acids
• surfactants for example Polysorbate 80
• sugars or polyalcohols for example sucrose or mannitol
• the present invention discloses a method for minimisation of dimer formation or formation of HMWS of a protein or other biological molecules, particularly of such molecules used in therapy, by putting the protein in a formulation of certain pH with minimal ionic strength, such as less than 30 mM, preferably less than 15 mM, most preferably less than 10 mM.
• a formulation of certain pH with minimal ionic strength such as less than 30 mM, preferably less than 15 mM, most preferably less than 10 mM.
• an aqueous composition comprises a protein or other biological molecule at a pH adjusted to a particular value, with reduced rate of dimer formation or formation of HMWS at such pH, further characterised in that the ionic strength of the composition is less than 30 mM, preferably less than 15 mM, most preferably less than 10 mM.
• the osmolarity of such composition can be adjusted to a required level using non-ionic species such as sugars or sugar alcohols.
• Some concentration of ionic species is typically needed as buffers in a formulation of a therapeutic protein. Therefore, the present invention may pose problems in ensuring sufficient buffering capacity whilst minimising the rate of aggregation. Such problems may be addressed by a specific choice of ionic species as buffers as follows: Since the ionic strength of an ionic species is proportional to the square of the charge of such species multivalent ions contribute considerably more strongly to ionic strength than monovalent ones. The use of monovalent ions as buffers is therefore preferable over the multivalent ones to ensure a degree of buffering capacity while minimising the ionic strength of the composition.
• an aqueous composition comprises a protein or other biological molecule at a pH adjusted to a particular value, with reduced rate of dimer formation or formation of HMWS at such pH, further characterised in that the composition is substantially free of multivalent ions and the ionic strength of the composition is less than 30 mM, preferably less than 15 mM, most preferably less than 10 mM.
• the osmolarity of such composition can be adjusted to a required level using non-ionic species such as sugars or sugar alcohols.
• amphiphilic compound comprises a considerable non-polar (hydrophobic) region such as a benzene nucleus or an aliphatic chain of four or more carbon atoms.
• the use of such amphiphilic compound further reduces the rate of dimer formation or formation of HMWS.
• the preferred example of such amphiphilic compound that can be usefully employed in the protein compositions according to the present invention is benzoic acid, particularly its ionic form (benzoate ion). Benzoic acid comprises one carboxylic group, which is predominantly charged at pH > 4.2, and a non-polar benzene nucleus. It is also an approved excipient in therapeutic formulations.
• an aqueous system comprises a protein or other biological molecule at a pH adjusted to a particular value, with reduced rate of dimer formation or formation of HMWS at such pH, further characterised in that (i) the ionic strength of the composition is less than 30 mM, preferably less than 15 mM, most preferably less than 10 mM, and (ii) the composition comprises a charged compound which contains an extensive hydrophobic region such as a benzene nucleus or an aliphatic chain of four or more carbon atoms. Benzoate ion is the preferred excipient in such composition.
• the osmolarity of such composition can be adjusted to a required level using uncharged species such as sugars or sugar alcohols.
• the present invention discloses a method for minimisation of dimer formation or formation of HMWS of a protein or other biological molecule by (i) putting the protein in a formulation of certain pH with minimal ionic strength, such as less than 30 mM, preferably less than 15 mM, most preferably less than 10 mM, and (ii) adding to the composition an ionic compound which contains an extensive hydrophobic region such as a benzene nucleus or an aliphatic chain of four or more carbon atoms.
• a formulation of certain pH with minimal ionic strength such as less than 30 mM, preferably less than 15 mM, most preferably less than 10 mM
• adding to the composition an ionic compound which contains an extensive hydrophobic region such as a benzene nucleus or an aliphatic chain of four or more carbon atoms.
• the present invention also addresses the stability of therapeutic molecules by reducing the rate of hydrolytic processes, such as cleavage of amide bonds or ester bonds.
• Hydrolysis is a particular stability problem of many different classes of molecules, ranging from small molecules to complex supramolecular systems.
• Examples of such undesirable processes include cleavage of a polysaccharide moiety from a carrier protein in a number of polysaccharide-based vaccines (e.g.
• Haemophilus influenzae b vaccine or a cleavage between key domains of fusion proteins (e.g. Etanercept). Acid or base hydrolysis is typically the mechanism of such degradation processes.
• Hydrolysis can also be part of the mechanism of more complex processes, such as deamidation of asparagine or aspartate isomerisation.
• the present invention is therefore also applicable in stabilising various molecules with respect to such processes which comprise hydrolysis as part of their molecular mechanism .
• Hydrolysis is a chemical reaction during which water molecule is split into hydrogen and hydroxide ions which go on to participate in cleavage of a particular covalent bond.
• Hydrolysis is known to be a very pH dependent process.
• proton transfer from molecules other than water can also be involved in the mechanism of hydrolytical cleavage.
• Hydrolysis is generally known to be strongly dependent on pH. Optimization of pH is therefore essential in order to reduce the rate of hydrolysis.
• other formulation parameters can bring about further reduction in the rate of hydrolysis.
• the present invention addresses additional key formulation parameters that can be applied to reduce further the rate of hydrolytic processes in formulations of a wide range of molecules and more complex systems.
• Another preferred feature of the present invention in relation to reducing the rate of hydrolysis is in combining the following formulation features in the formulation of a particular molecule or a more complex system: •
• Minimal ionic strength ionic strength of the formulation is kept minimal, such as less than 40 mM, preferably less than 20 mM, most preferably less than 1O mM.
• the formulation is substantially free of a conventional buffer, i.e a compound with pK a within 1 unit of the pH of the composition at the intended temperature range of storage of the composition, and comprises one or more additives (displaced buffers) which are capable of exchanging protons with other molecules and have pK a values at least 1 unit more or less than the pH of the composition at the intended temperature range of storage of the composition; the art of applying displaced buffers to formulations of biologicals is described in WO2008/084237.
• the rate of the undesirable hydrolytic process can be reduced substantially.
• the formulation is kept at a pH at which the rate of hydrolysis is minimal.
• Optimal pH can be established experimentally.
• the invention is applicable at pH away for such pH optimum.
• the present invention discloses a method for minimisation of the rate of hydrolytic process on a molecule or supramolecular system by putting the molecule or the system in a formulation of certain pH with minimal ionic strength, such as less than 40 mM, preferably less than 20 mM, most preferably less than 10 mM.
• the pH of the composition is adjusted to a level at which the rate of the undesirable hydrolytic process is minimal.
• an aqueous composition comprises a molecule or a supramolecular system, at a pH adjusted to a particular value, further characterised in that the ionic strength of the composition is less than
• the osmolarity of such composition can be adjusted to a required level using non-ionic species such as sugars or sugar alcohols.
• the pH of the composition is adjusted to a level at which the rate of the undesirable hydrolytic process is minimal.
• an aqueous composition comprises a molecule or a supramolecular system, at a pH adjusted to a particular value, further characterised in that the composition is substantially free of multivalent ions and the ionic strength of the composition is less than 40 mM, preferably less than 20 mM, most preferably less than 10 mM.
• the osmolarity of such composition can be adjusted to a required level using non-ionic species such as sugars or sugar alcohols.
• the pH of the composition is adjusted to a level at which the rate of the undesirable hydrolytic process is minimal.
• an aqueous composition comprises a molecule or supramolecular system, at a pH adjusted to a particular value, further characterised in that the composition is substantially free of conventional buffer and comprises one or more additives which are capable of exchanging protons with the protein and have pK a values at least 1 unit more or less than the pH of the composition at the intended temperature range of storage of the composition; the ionic strength of the composition is less than 40 mM, preferably less than 20 mM, most preferably less than 10 mM.
• the osmolarity of such composition can be adjusted to a required level using non-ionic species such as sugars or sugar alcohols.
• the pH of the composition is adjusted to a level at which the rate of the undesirable hydrolytic process is minimal.
• Various hydrolytic processes are catalysed by proton transfer at the cleavage site facilitated by molecules other than water, for example molecules of buffers. Without wishing to be bound by theory, it is believed that the benefit of using displaced buffers instead of conventional buffers in compositions of molecules that are prone to hydrolytic cleavage is in minimising the rate of proton transfer from molecules of conventional buffers to or from the cleavage site.
• Example 1 The invention is illustrated by the following Examples: Example 1
• HMWS HMWS was followed in a solution of alpha-glucosidase (12.5 mg/mL) using the following size-exclusion chromatographic method:
• the mobile phase was 25 mM sodium phosphate (pH 6.2) containing 150 mM NaCI.
• the mobile phase was filtered prior to its use.
• the liquid chromatograph (Agilent 1100 series) was equipped with a 214 nm detector, guard column and a 7.8 * 300 mm BioSep SEC-S2000 column. The flow rate was maintained at 0.45 mL/min. 20 ⁇ l_ of aqueous samples of alpha-glucosidase were injected.
• the level of high molecular weight species was expressed as the percentage of the total peak area of all peaks with elution time shorter than that of the main peak versus the area of the main peak.
• the aggregation rate was studied at 25 0 C in the presence of 4 mM TRIS buffer.
• the buffering strength of TRIS buffer at pH ⁇ 6.5 is minimal, but sufficient buffering capacity originated from the relatively concentrated enzyme itself at such pH.
• the optimum pH with respect to minimal formation of HMWS was found to be around 6.5.
• the aggregation rate was higher both at lower and at higher pH.
• Increase in ionic strength resulted in considerable increase in the rate of HMWS, especially outside the optimum pH (Table 1). So, whilst the increase in ionic strength resulted only in moderate increase of the aggregation rate at ph 6.5 the increase was considerably higher both at higher and at lower pH.
• HMWS Formation of HMWS (%) at 25 0 C (3 weeks) in aqueous formulation of alpha- glucosidase (12.5 mg/mL) in the presence of 4 mM TRIS buffer and the indicated concentration of NaCI.
• HMWS Formation of HMWS (%) at 25 0 C (4 weeks) in aqueous formulation of alpha- glucosidase (12.5 mg/mL) in the presence of TRIS buffer (2 mM) either in the presence or in the absence of benzoic acid (2 mM).
• HMWS Formation of HMWS (%) at 4O 0 C (2 weeks) in aqueous formulation of alpha- glucosidase (12.5 mg/mL) in the presence of TRIS buffer (2 mM) either in the presence or in the absence of benzoic acid (2 mM).
• Hydrolysis of the polysaccharide antigen (polyribose-phosphate-ribose, PRP) from a carrier protein is a particular problem of the Haemophilus influenzae b (Hib) vaccine.
• the extent of the hydrolysis can be expressed in terms of the percentage of the free (i.e. unbound to the carrier protein) PRP in the formulation.
• the method is based on separation of free PRP from bound PRP and subsequent quantification of PRP in both fractions by the Bial reaction (Kabat EA, Mayer M: Carbohydrate estimation. In: Experimental immunochemistry. Springfield, IL: C Thomas, 1961. p. 526-37).
• the free PRP was separated from the bound PRP by precipitating Hib vaccine with deoxycholate.
• Neat sample 200 ⁇ l; at 50 ⁇ g/ml was pipetted in to a microcentrifuge tube.
• 200 ⁇ l analytical water was used.
• deoxycholate (0.1% w/v) was added and then vortexed. These were then incubated (30 mins, at +4 0 C).
• hydrochloric acid 50 ⁇ l, 1M was added to each tube. All tubes were vortexed and then centrifuged (45mins at 5.2g) at +4 0 C.
• pellet solutions were now ready for analysis of ribose bound to glycoprotein.
• ferric chloride in 10 M hydrochloric acid.
• 20 ⁇ l orcinol dye (10% in absolute ethanol) solution was added in the mixture incubated at 95 0 C for 40 min.
• the tubes were cooled in a beaker of cold water (+4 0 C).
• the contents were transferred to cuvettes and the absorbance at 670 nm was measured. The values obtained must have the appropriate blank values subtracted.
• HibTITER for example HibTITER, Wyeth
• HibTITER for example HibTITER, Wyeth
• saline as the key formulation ingredient. It was shown that a significant improvement in stability can be achieved if the vaccine is formulated in a low ionic strength environment at pH around 6. Histidine was used a buffer in this case and uncharged 1 ,2-propanediol was used as tonicity modifier. Stability was studied at 4O 0 C. More than 20% increase of free PRP can be observed in the currently marketed formulation (saline, pH ⁇ 6) of Hib vaccine after 3 weeks and >60% after 13 weeks of incubation at 4O 0 C.

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